Preparing membrane receptors from extracellular baculoviruses

Abstract

The present invention describes a method for producing membrane receptors based on a baculovirus/insect cell system. The invention further includes the receptors produced by the method described herein, a model for studying the properties of these membrane receptors and an assay to screen molecules that are active on these membrane receptors.

1. A method of determining whether a membrane receptor present in recombinant extracellular baculoviruses is active, the method comprising:
 providing recombinant extracellular baculoviruses in which the membrane receptor is present; 
 contacting the extracellular baculoviruses with a ligand to the membrane receptor; and 
 determining whether binding of the ligand to the receptor occurs, wherein said binding is an indication that the receptor is active. 
 
     
     
       2. A method as defined in  claim 1 , wherein said receptor is a G-protein-coupled receptor. 
     
     
       3. A method as defined in  claim 2 , wherein said G-protein-coupled receptor is β2AR, M1-muscarinic receptor (M1-R) or D1-dopaminergic receptor (D1-R). 
     
     
       4. A method as described in  claim 3 , wherein said ligand is [ 125 I]ICYP, [3H]pirenzepine or [ 125 I]-R(+)SCH23390. 
     
     
       5. A method of determining whether a membrane receptor from isolated membranes is in active form, wherein said membrane receptor is produced by infecting a culture of insect cells with recombinant baculoviruses encoding the gene for said membrane receptor and said membrane receptor is harvested from extracellular baculoviruses generated by said insect cells, the method comprising:
 contacting the receptor with a ligand; and 
 determining whether binding of the ligand to the receptor occurs, wherein said binding is an indication that the receptor is active. 
 
     
     
       6. A method as defined in  claim 5 , wherein said receptor is a G-protein-coupled receptor. 
     
     
       7. A method as defined in  claim 6 , wherein said G-protein-coupled receptor is β2AR, M1-muscarinic receptor (M1-R) or D1-dopaminergic receptor (D1-R). 
     
     
       8. A method as described in  claim 7 , wherein said ligand is [ 125 I]ICYP, [3H]pirenzepine or [ 125 I]-R(+)SCH23390. 
     
     
       9. A method of screening for molecules which are active on a membrane receptor present in recombinant extracellular baculoviruses, the method comprising:
 providing recombinant extracellular baculoviruses in which the membrane receptor is present; 
 contacting the extracellular baculoviruses with a test molecule and a ligand to the membrane receptor; and 
 determining whether binding of the ligand is decreased in the presence of the test molecule, wherein a decrease in said binding is an indication that the test molecule competes with the ligand for the receptor. 
 
     
     
       10. A method as defined in  claim 9 , wherein said receptor is a G-protein-coupled receptor. 
     
     
       11. A method as defined in  claim 10 , wherein said G-protein-coupled receptor is β2AR, M1-muscarinic receptor (M1-R) or D1-dopaminergic receptor (D1-R). 
     
     
       12. A method as described in  claim 11 , wherein said ligand is [ 125 I]ICYP, [3H]pirenzepine or [ 125I ]-R(+)SCH23390. 
     
     
       13. A method of screening for molecules which are active on a membrane receptor from isolated membranes, wherein said membrane receptor is produced by infecting a culture of insect cells with recombinant baculoviruses encoding the gene for said membrane receptor and said membrane receptor is harvested from extracellular baculoviruses generated by said insect cells, the method comprising:
 contacting the receptor with a test molecule and a ligand; and 
 determining whether binding of the ligand is decreased in the presence of the test molecule, wherein a decrease in said binding is an indication that the test molecule competes with the ligand for the receptor. 
 
     
     
       14. A method as defined in  claim 13 , wherein said receptor is a G-protein-coupled receptor. 
     
     
       15. A method as defined in  claim 14 , wherein said G-protein-coupled receptor is β2AR, M1-muscarinic receptor (M1-R) or D1-dopaminergic receptor (D1-R). 
     
     
       16. A method as described in  claim 15 , wherein said ligand is [ 125 I]ICYP, [3H]pirenzepine or [ 125 I]-R(+)SCH23390. 
     
     
       17. A method of determining whether two or more membrane receptors are present in recombinant extracellular baculoviruses in active form, the method comprising:
 providing recombinant extracellular baculoviruses in which two or more membrane receptors are present; 
 contacting the extracellular baculoviruses with a ligand specific to each membrane receptor; and 
 determining whether binding of each ligand to its receptor occurs, wherein said binding is an indication that the receptor is active. 
 
     
     
       18. A method as defined in  claim 17 , wherein said receptors are G-protein-coupled receptors. 
     
     
       19. A method as defined in  claim 18 , wherein said G-protein-coupled receptors include at least two of β2AR, M1-muscarinic receptor (M1-R) and D1-dopaminergic receptor (D1-R). 
     
     
       20. A method as described in  claim 17 , wherein said receptors and ligands are chosen from the following: β2AR and [ 125 I]ICYP, M1-muscarinic receptor (M1-R) and [3H]pirenzepine, and D1-dopaminergic receptor (D1-R) and [ 125 I]-R(+)SCH23390. 
     
     
       21. A method of determining whether two or more membrane receptors from isolated membranes derived from recombinant extracellular baculoviruses are in active form, the method comprising:
 contacting the membrane receptors with ligands known to be specific to each one of the membrane receptors; and 
 determining whether binding of the ligands to their respective membrane receptors occurs, wherein said binding is an indication that the membrane receptors are active. 
 
     
     
       22. A method as defined in  claim 21 , wherein said receptors are G-protein-coupled receptors. 
     
     
       23. A method as defined in  claim 22 , wherein said G-protein-coupled receptors include at least two of β2AR, M1-muscarinic receptor (M1-R) and D1-dopaminergic receptor (D1-R). 
     
     
       24. A method as described in  claim 23 , wherein said receptors and ligands are chosen from the following: β2AR and [ 125 I]ICYP, M1-muscarinic receptor (M1-R) and [3H]pirenzepine, and D1-dopaminergic receptor (D1-R) and [ 125 I]-R(+)SCH23390.